title: Experimental platform to study heavy metal ion-enzyme interactions and amperometric inhibitive assay of Ag+ based on solution state and immobilized glucose oxidase.
abstract: The heavy metal (HM) ion-enzyme interaction is an important research topic in many areas. Using glucose oxidase (GOx) as an example, a comprehensive experimental platform based on quartz crystal microbalance and electroanalysis techniques is developed here to quantitatively study the HM ion-enzyme interactions and amperometric inhibitive assays of HM ions. The effects of some common HM ions on the bioactivities of solution-state GOx (GOx(s)), electrode surface-adsorbed GOx (GOx(ads)), and polymer-entrapped GOx (GOx(e)) are comparatively examined on the basis of anodic amperometric detection of enzymatically generated H(2)O(2). Ag(+) shows the strongest inhibition effect among the HM ions examined, and the inhibitive assays of Ag(+) based on GOx(s), GOx(ads), and GOx(e) entrapped in poly(l-noradrenalin) (PNA) give limits of detection (LOD) of 2.0, 8.0, and 5.0 nM (S/N = 3), respectively. Inhibition effects of Hg(2+), Cu(2+), and Co(2+) are detectable only at 15 μM or higher concentrations, and the other HM ions show undetectable inhibition even at 1.0 mM. The developed experimental platform allows one to quantify the number of the bound HM ions per GOx(ads) molecule at various inhibition percentages. In addition, the electrosynthesized PNA matrix to entrap GOx for an inhibitive assay of Ag(+) shows the lowest competitive affinity to HM ions and gives the highest sensitivity, as compared with several other polymer matrixes commonly used for the inhibitive assay. The suggested experimental platform is recommended for wide applications in enzymatic inhibitive assays and quantitative studies of the inhibition effects of HM ions on many other redox-event-relevant enzymes.
authors.affiliation: Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, P. R. China.
authors.forename: Chao
authors.initials: C
authors.lastname: Chen
authors.forename: Qingji
authors.initials: Q
authors.lastname: Xie
authors.forename: Lihua
authors.initials: L
authors.lastname: Wang
authors.forename: Cong
authors.initials: C
authors.lastname: Qin
authors.forename: Fangyun
authors.initials: F
authors.lastname: Xie
authors.forename: Shouzhuo
authors.initials: S
authors.lastname: Yao
authors.forename: Jinhua
authors.initials: J
authors.lastname: Chen
mesh_terms: D001667:Binding, Competitive; D004563:Electrochemistry / Q000379:methods*; D004566:Electrodes; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D004800:Enzymes, Immobilized / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D005949:Glucose Oxidase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D011108:Polymers / Q000737:chemistry; D011485:Protein Binding; D012834:Silver / Q000378:metabolism* / Q000494:pharmacology*; D012996:Solutions
chemical_list: D004791:Enzyme Inhibitors; D004800:Enzymes, Immobilized; D011108:Polymers; D012996:Solutions; D012834:Silver; D006861:Hydrogen Peroxide; D005949:Glucose Oxidase